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CHRONIC CANNULATION OF THE INTRADURAL OR EXTRADURAL SPACE IN THE RAT
Br. J. Anaesth. (1984), 56,405
CHRONIC CANNULATION OF THE INTRADURAL OR EXTRADURAL SPACE IN THE RAT M. BAHAR, M. ROSEN AND M. D. VICKERS
In recent years a variety of drugs have been administered to the extradural and intrathecal spaces in man. Although, ideally, such drugs should be tested first in animals., spinal injection in conscious animals has not been performed frequently because the use of large animals such as dog, sheep, cat and primate, is restricted by regulatory and financial considerations. Nevertheless, two methods have been described. Chronic cannulation of the intrathecal space in the rat has been carried out by introducing a polyethylene catheter into the junction of the first cervical vertebra and the occipital bone (cisternal membrane), and then passing the catheter distally between the spinal cord and dura (Yaksh and Rudy, 1976). However, these authors were unable to demonstrate the spread of narcotics centrally, possibly because the catheter obstructed the intradural space from the insertion point to the tip of the cannula at the lumbar enlargement (Bahar, Orr and Dundee, 1981). Permanent motor paresis developed in as many as 10-15% of rats (Yaksh and Rudy, 1976). An alternative intradural approach through the lumbar region has been used but published only in abstract (Kim and Stoelting, 1980). Extradural cannulation was described first by van den Hoogen and Colpaert (1981), who drilled a hole in the lamina of a
M.
BAHAR, M.D.; M.
ROSEN,* F.F.A.ILCS.; M.
D.
VICKERS,
F.F-A.R.C.S.; Department of Anaesthetics, Welsh National School of Medicine, Heath Park, Cardiff CF4 4XN. •Present address, for correspondence: Department of Anaesthetics, University Hospital of Wales, Heath Park, Cardiff CF4 4XW.
Downloaded from http://bja.oxfordjournals.org/ at New York University on May 5, 2015
An animal preparation has been developed to test therapeutic agents in the extradural and intradural spaces. Under anaesthesia a hole was drilled in the penultimate lumbar vertebra of male Wistar rats and the appropriate space cannulated. The catheter was tunnelled subcutaneously to emerge at the neck. There was no spinal cord or meningeal reaction after 1 month. Catheters remained patent for 3 months. The method of cannulation allowed free rostral spread of drugs. Using plain 2% lignocaine, paralysis and anaesthesia of the hind limbs required an intradural volume of 32 ± 3 ulitre: the required volume on extradural injection was 46±2nlitre (P<0.01). Paralysis of all limbs required an intradural volume of 115± 12ulitre. Respiratory arrest and death required a mean intradural volume of 179±15filitre.
lumbar vertebra. Extradural cannulation has also been performed in the mid-thoracic region, but the method of cannulation was not described (Eimerl et al., 1983). As central spread of drugs is of considerable concern when administered by either route, the technique described here has been developed. It offers a choice of intradural or extradural cannulation in the lumbar region of the rat without obstructing the spinal canal above the site of injection. The technique can be used to demonstrate segmental analgesia and the complications occasioned by the central spread of the drug. THE PREPARATION
The catheters (tig. 1) Nylon catheters, o.d. 0.75mm and i.d. 0.25mm (Portex Ltd), were cut in 200-mm lengths. The deadspace was decreased by inserting 190 mm of a 4/0 nylon surgical suture into the lumen. The terminal 10 mm were left unfilled to allow a 30-s.w.g. needle to be connected. The deadspace was measured and was usually between 3.8 and 4.5 ftlitre, and never more than 5/ilitre. Two 5/0 surgical nylon sutures were glued to the catheter with epoxy resin, 25 mm from the inserted end and 50 mm from the exterior end. The end of the catheter to be inserted was marked at 5-mm intervals for 25mm. The catheter was flushed with air and the exterior end sealed with epoxy resin. Catheters were packed individually and sterilized with ethylene oxide. Anaesthesia Anaesthesia was induced in male Wistar rats (270-400 g in weight) by placing them in a closed © The Macmillan Press Ltd 1984
BRITISH JOURNAL OF ANAESTHESIA
406
box containing 5% halothane in oxygen. Anaesthesia was maintained via a loose-fitting, coneshaped mask through which a flow of 0.2 litre"1 min of 2.5% halothane in oxygen was passed.
dura. Occasionally, resistance was felt as the tip met the lamina of the next vertebra. The catheter was sutured to the overlying lumbar fascia which was then closed with interrupted 5/0 nylon sutures. The catheter was exteriorized through a second skin incision in the midline over the upper cervical area Surgical procedure With the rat in the prone position, the back was by tunnelling it under the skin. The cervical portion shaved and a line joining the iliac crests defined. The of the catheter was fixed to the fascia of the neck skin was incised for 4 cm in a cervical and 1 cm in a muscles with a 5/0 nylon suture and the skin incicaudal direction, in the midline from this line. The sions closed with interrupted 4/0 nylon. fascia covering the lumbar muscles was divided in the same direction as the skin incision, 1 mm lateral Injection apparatus to the midline on the side nearest to the operator. Hamilton 50-^litre and 100-nlitre gas-tight Through this opening, the lamina of the penulti- syringes were used with 28-gauge needles. The mate vertebra (usually 1 cm rostral to the iliac crests) syringe was attached to a 200-mm length of was exposed by blunt dissection. A dissecting mic- polythene tubing (0.28 mm i.d. 0.61mm o.d.), roscope was used at 16 x magnification and a 1-mm and this was joined to the implanted catheter by a hole drilled with a ball-shaped diamond drill (1 mm) double-ended 30-s.w.g. dental needle. The whole in the mid-point of the lamina. When spinal vessels assembly was sterilized with ethylene oxide. It was overlying the dura were identified through the thin- primed with the drug to be injected before being ned bone, drilling was stopped and the bone flap connected to the implanted catheter. Before the first pierced with a fine hook, bone fragments being injection the sealed tip was cut off and 5nlitre of elevated and the dura exposed. For intradural can- solution injected with a 1-mm bubble of air to nulation the dura was tensed and torn gently with uncover any leaks, and demonstrate the patency of fine-pointed (0.15-mm) forceps until CSF leaked the catheter. Subsequently, the catheter could be out and the cauda equina was seen to bulge. The left open for up to 3 months. The catheter was catheter was inserted for 10-15 mm in a cervical flushed with physiological saline after each experidirection and usually slid in easily. For extradural ment (with four times the deadspace volume cannulation the catheter was fed in a cervical direc- (20nlitre)) using a clean syringe system. Persistent tion for a similar distance outside the unopened separation of the needle tubing from the implanted
Downloaded from http://bja.oxfordjournals.org/ at New York University on May 5, 2015
FlG. 1. The catheter for implantation: 1 •* the exteriorized tip is glued with epory resin; 2— points at which sutures are glued to catheter; 3-markings at 0.5-cm intervals; 4-monofilamcnt 5/0 nylon suture with threaded round-bodied needle; 5— region where the internal filament is absent; 6= the nylon catheter with the internal filament
CHRONIC SPINAL CANNULATION IN THE RAT catheter on injection indicated that the catheter had become blocked; this could be avoided by flushing the catheter at least twice a week. EXPERIMENTAL METHODS
dose to six rats, (b) In six rats, morphine 20 mg kg" 1 was administered i.p. Twenty-five minutes later, when analgesia was well-established, naloxone 5 ulitre (0.4 mg ml"1) was injected to the intrathecal space. Evaluation of analgesia and muscle power. The extent of segmental analgesia was evaluated using the haemostat pinch test; analgesia was graded as present or absent. Muscle power was graded as normal or impaired, the latter when the affected limbs were not functioning normally. Downloaded from http://bja.oxfordjournals.org/ at New York University on May 5, 2015
Injections Injections were performed at least 24 h after recovery from surgery, recovery being defined as the ability to walk steadily, drink and feed. The rat was restrained whilst the injection system was connected, but was then free to move around its cage while injections were made. The acceptability of the position of the catheter was determined by an injection of 2% lignocaine plain (preservative free). If 35 ulitre into the intradural or 50 ulitre into the extradural space did not anaesthetize the rear limbs, the animal was excluded. This happened occasionally, especially in rats which had been catheterized for more than 4 weeks. In those cases an injection of methylene blue always showed a leak from the catheter site.
407
Histopathology. Immediately following sacrifice, 10% formalin saline solution 500 ulitre was injected through the implanted catheter. The whole spinal column was removed and fixed in the same solution for 3 days. It was then decalcified for 2 weeks. Histological sections were stained with haematoxylin and eosin. RESULTS
Evaluation of the model
Evaluation of model
Speed of onset of intradural blockade. Twenty-five microlitre of 2% lignocaine was injected into six rats over 3-4 s and the time noted until the rear limbs dragged.
Speed of onset. The onset of intradural block took 6.3±0.6s.
Equipotent volumes and doses for various levels of block. The mean volume which anaesthetized and Volumes required for equipotent intradural and ex- paralysed the rear limbs after intradural injection tradural blockade. Ten-microlitre increments of 2% were 32 /ilitre, and 46 ^litre after extradural injeclignocaine were injected every minute at 1 ulitre s~' tion ( P < 0 . 0 1 ) . Recovery occurred in a mean time in a total of 23 (11 intradural and 12 extradural) rats of 43 min after intradural and in 57 min following until both rear limbs dragged and there was no extradural injection ( P < 0 . 0 5 ) (tableI). The mean response to a pinch with a haemostat in the lumbar volume of intradural injection which anaesthetized area. Recovery of motor and sensory function was all the limbs was 115 filitre and the mean volume of timed. intradural injection which resulted in respiratory arrest was 179 ^litre (table II). Volume of intradural injection to anaesthetize all limbs. In six rats, increments of 2% lignocaine 25 ulitre were injected every minute until the rat could not support its weight; the abdomen and chest TABLEI. Relative volumes (meant ±SEM) of2% lignocaitu given by rested on the floor of the cage. Recovery to normal extradural and intradural injtaion required to anaesthetize the rear function was timed. limbs Volume of intradural injection to cause respiratory arrest. In five rats, an initial injection of 2% lignocaine 50 ulitre was followed by increments of 25 ulitre every minute until respiration ceased. Effects of morphine, (a) Preservative-free morphine 45filitre (1.5mgml~ l ) was given in a single
Intradural injection ( » - 12) Eitradural injection (n-11)
Weight (g)
2%lignocaine (ulitre)
Recovery (min)
328±11
32±3
43±3
312±7
46±2
57±7
BRITISH JOURNAL OF ANAESTHESIA
408 TABLE II. Intradural volume (mean + SEM) oflignocaine necessary (a) to anaesthetize all limbs; (b) to cause respiratory arrest Weight (g)
2%Lignocaine (filitre)
Recovery (min)
321±9
115+12
63±4
323 ±14
179 ±10
Death
Four limb anaesthesia
(n-6) Respiratory arrest (it = 5)
Histopathology. Serial sections were made of the spinal cord in three rats which had received no drugs. This revealed no reaction to the catheter (fig. 2). The hole in the lamina showed new bone formation surrounding the catheter. There was
DISCUSSION
Local anaesthetics are used widely via the extradural, or intradural, routes and neurotoxicity is sometimes suspected. In addition opioid drugs have been administered by these routes in man (Christensen and Anderson, 1982; Cooper et al., 1982; Budd, Robson and Brown, 1983). Thus far, no formal screening procedure has emerged, probably, because of difficulties in developing an acceptable animal model which allows central spread of drugs. The method described by Yaksh and Rudy (1976), although extremely valuable, has only been described in rats for cannulation of the intradural space. However, on occasions, as the cannula passed distally from the cisternal membrane, major cord damage resulted, either immediately or later (Yaksh and Rudy, 1977) possibly from movements of the head transmitted to the tip, because the catheter is fixed to the skull. Furthermore, with the cranial approach, the central complications which have been seen in man were not observed (Yaksh and
w FIG. 2. Transvene tection of the spinal cord at mid lumbar region: 1 — Intradural catheter between cauda equina nerves.
Downloaded from http://bja.oxfordjournals.org/ at New York University on May 5, 2015
Effects of morphine, (a) The intrathecal injection of morphine produced analgesia in the head, and all four limbs; in two rats there was no analgesia in the tail, (b) Intrathecal naloxone reversed the analgesic effects of intraperitoneal morphine in all animals; in limbs, head and tail.
minor leucocyte infiltration and giant cell formation in the muscles and subcutaneous tissues.
CHRONIC SPINAL CANNULATION IN THE RAT Imrathecal cannulation from the lumbar route
409 Intrathecal cannutauon foam cervical region lYaksh and Rudy 19771
25 rrun after intrathecal morphine 45 plitre 1 l
25 mm after morphine 20 mg kg" 1 i p
FIG. 3. Comparison of the behaviour of the lumbar cannulation model with the cervical approach of Yaksh and Rudy (1977). After cervical cannulation, analgesia did not occur in the head and nalozone failed to reverse the systemic effects of interperitoneal morphine in the head. Neither of these features are found after lumbar cannulation, although one-third of the rats failed to develop analgesia in the tail.
Rudy, 1977) suggesting that there must have been some obstruction to central spread (fig. 3). In contrast, the technique described here not only allows either extradural or intradural cannulation, but central effects of the drugs were produced readily. Respiratory arrest was induced with lignocaine and with opioids (unpublished observations), and the central effects of systemically administered morphine were antagonized by the spinal administration of naloxone (fig. 3). The model has also been used successfully to demonstrate differential sensory blockade without motor paresis using intrathecal cocaine (Bahar et al., 1984). Furthermore, neurological damage has been uncommon ( < 3% in more than 200 cannulations to date), and if it occurs it is obvious immediately. Finally, recovery appeared to be slower with Yaksh and Rudy's technique since subcutaneous fluids were infused for 24 h after surgery (Larsen and Christensen, 1982). In the method described here, full recovery occurred in most animals within 1 h. Whilst this may be a result of differences in anaesthetic technique, we believe that delay in recovery may be the result of medullary oedema. In summary, the rapidity of recovery, the low frequency of neurological damage, the demonstra-
tion of central spread of drugs and the possibility of Ijpth extradural and intradural cannulation make lumbar cannulation of value for research into the effects of drugs administered in the extradural and intradural spaces. ACKNOWLEDGEMENTS
We acknowledge in particular, the help of Mr D. Ritchie, Head of the Animal Unit; the Department of Pharmacology and the Pharmacy, University Hospital of Wales; the Department of Medical Illustration; the secretarial staff of the Department of Anaesthetics and Mrs J. Brown and Mr C. Juniper for their valuable technical assistance. REFERENCES
Bahar, M., Nunn, J. F., Rosen, M., and FleckneU, P. (1984). Differential sensory and motor blockade after spinal cocaine in the rat and marmoset. Eur. J. Anaathaiol., (in press). Orr, I. A., and Dundee, J. W. (1981). Central action of spinal opiates. Anathesiology, 55, 334. Budd, K., Robson, P. J., and Brown, P. M. (1983). The treatment of chronic pain by the use of meptazinol administered into the epidural space. Postgrad. Med. J., 59,68. Christensen, F. R., and Andersen, L. W. (1982). Adverse reaction to extradural buprenorphine. Br. J. Anaath., 54,476. Cooper, G. M., Goodman, N. W., Prys Roberts, C , Jacobson, L., and Douglas, G. (1982). Ventilatory effects of extradural diamorphine. Br. J. Anaath., 54,239. Eimerl, D., Papir-Kricheli, D., Evron, S., and Cannon, A.
Downloaded from http://bja.oxfordjournals.org/ at New York University on May 5, 2015
5 mm after intrathecal nakwone 5ug
BRITISH JOURNAL OF ANAESTHESIA
410
LANGDAUERNDE KANNULIERUNG DES INTRADURAL-UND EXTRADURALRAUMS BEI DER RATTE
(1983). Experimental epidural analgesia: Direct effect of morphine at the spinal level; in Advances in Pain Research and Therapy Volume 5 (eds J. J. Bonica, U. lindblom and A. Iggo), p. 487. New York: Raven Press. van den Hoogen, R. H., and Colpaert, F. C. (1981). Long-term catheterization of the lumbar epidural space in rats. Pharmacol. Biochtm. Bthav., 15, 515. Kim, K.C., and Stoelting, R. K. (1980). Effect of droperidol on the duration of analgesia and development of tolerance. Antsthesiology, 53, S219. Larsen, J., and Christensen, A. V. (1982). Subarachnoidal administration of the 5-HT uptake inhibitor citalopram points to the spinal role of 5-HT in morphine antinociception. Pain, 14, 339. Yaksh, T. L., and Rudy.T. A. (1976). Chroniccatheterisationof the spinal subarachnoid space. Physiol. Bthav., 17, 1031. (1977). Studies on the direct spinal action of narcotics in the production of analgesia in the rat. / . Pharmacol. Exp. Thtr., 202,411.
Im Tierversuch wurden therapeutische Medikamente im Eitraduralraum und im Intradurah*aum getcstet. Bei mannlichen Wistar-Ratten wurde in Narkose ein Loch in den vorletzten Lendenwirbel gebohrt, durch welches ein Katheter geschoben wurde. Der Katheter wurde dann subkutan bis in den Nacken getunnelt. NacheinemMonatgabeswederimRuckenmarknoch an den Meningen eine Reizung. Die Katheter funktionierten drci Monate lang. Die Mcthode erlaubte eine freie rostrale Ausbreitung der Medikamente. Mit 2% igem Lignocain wurde zur T Jjhmnng und Anasthesie der Hinterbeine ein intradurales Volumen von 32±3fU benStigt; extradural waren 46±2(il notig (P<0,01). Zur T,ahnning allcr Bcine waren 115±12jil notig. Atemstillstand und Tod traten-nach einem mittleren intraduralen Volumen von 179 ± 15 fU ein.
CATHETERISME CHRONIQUE DE L'ESPACE SOUS-DURAL OU PERIDURAL CHEZ LE RAT
CANULACI6N CR6NICA DEL ESPACIO INTRADURAL O EXTRADURAL EN LA RATA
ZUSAMMENFASSUNG
SUMARIO
Se elaboro una preparacion animal para ensayar agentes terapeuticos en los espacios intradurales y extradurales. Bajo anestesia, se perforo un hueco en la penultima vertebra lumbar de ratas mafhaa Wistar y se canulo el espacio apropiado. Seatraveso horadando debajo de la piel una sonda para hacerla salir al cucUo. No hubo ningiinn rcaccion meningea ni tampoco de la medula espinal despues de 1 mes. Las sondas permanecieron pervias durante 3 meses. El metodo de canulacion permiti6 la libre diseminacion de las substancias por el rostro. Al usar lignocaina liana al 2%, se necesito para la paralisis y la anestesia dc los miembros traseros un volumen intradural de 32±3nlitro: el volumen necesario de inyeccion extradural era de 46±2nlitro (P<0,01). Para la paralisis de todos los miembros, se necesito un volumen intradural de 115 ± 12(xlitro. El paro respiratorio y la muerte exigieron un volumen intradural medio de 179 ± 15 ulitro.
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RESUME
Une preparation animale a etc mise au point pour tester des agents therapcutiques dans l'espace peridural et dans l'espace sousdural. Sous anesthesie generale, un trou etait perce dans l'avantdernicre vertebre lombaire de rats Wistar males, l'espace prcetabli etait catheterise et le catheter etait tunnelisc sous la pcau pour emerger au niveau du cou. Au bout d'un mois, il n'y avait ni reaction medullaire ni reaction meningee. Les catheters restaient permcables 3 mois. La technique de catheterisme permettait une libre diffusion des agents. Avec de la lignocaine a 2%, le bloc moteur et sensitif des panes arrieres demandait un volume sousarachnoidien de 32 ±3^1 et un volume peridural de 46±2(J CP<0,01). Un bloc moteur des quatre membres demandait un volume sous-arachnoidien de 115 ± 12 fi\. L'arrct respiratoire et la mort demandait un volume sous-arachnoidien moyen de 179±15nl.
CHRONIC CANNULATION OF THE INTRADURAL OR EXTRADURAL SPACE IN THE RAT M. BAHAR, M. ROSEN AND M. D. VICKERS
In recent years a variety of drugs have been administered to the extradural and intrathecal spaces in man. Although, ideally, such drugs should be tested first in animals., spinal injection in conscious animals has not been performed frequently because the use of large animals such as dog, sheep, cat and primate, is restricted by regulatory and financial considerations. Nevertheless, two methods have been described. Chronic cannulation of the intrathecal space in the rat has been carried out by introducing a polyethylene catheter into the junction of the first cervical vertebra and the occipital bone (cisternal membrane), and then passing the catheter distally between the spinal cord and dura (Yaksh and Rudy, 1976). However, these authors were unable to demonstrate the spread of narcotics centrally, possibly because the catheter obstructed the intradural space from the insertion point to the tip of the cannula at the lumbar enlargement (Bahar, Orr and Dundee, 1981). Permanent motor paresis developed in as many as 10-15% of rats (Yaksh and Rudy, 1976). An alternative intradural approach through the lumbar region has been used but published only in abstract (Kim and Stoelting, 1980). Extradural cannulation was described first by van den Hoogen and Colpaert (1981), who drilled a hole in the lamina of a
M.
BAHAR, M.D.; M.
ROSEN,* F.F.A.ILCS.; M.
D.
VICKERS,
F.F-A.R.C.S.; Department of Anaesthetics, Welsh National School of Medicine, Heath Park, Cardiff CF4 4XN. •Present address, for correspondence: Department of Anaesthetics, University Hospital of Wales, Heath Park, Cardiff CF4 4XW.
Downloaded from http://bja.oxfordjournals.org/ at New York University on May 5, 2015
An animal preparation has been developed to test therapeutic agents in the extradural and intradural spaces. Under anaesthesia a hole was drilled in the penultimate lumbar vertebra of male Wistar rats and the appropriate space cannulated. The catheter was tunnelled subcutaneously to emerge at the neck. There was no spinal cord or meningeal reaction after 1 month. Catheters remained patent for 3 months. The method of cannulation allowed free rostral spread of drugs. Using plain 2% lignocaine, paralysis and anaesthesia of the hind limbs required an intradural volume of 32 ± 3 ulitre: the required volume on extradural injection was 46±2nlitre (P<0.01). Paralysis of all limbs required an intradural volume of 115± 12ulitre. Respiratory arrest and death required a mean intradural volume of 179±15filitre.
lumbar vertebra. Extradural cannulation has also been performed in the mid-thoracic region, but the method of cannulation was not described (Eimerl et al., 1983). As central spread of drugs is of considerable concern when administered by either route, the technique described here has been developed. It offers a choice of intradural or extradural cannulation in the lumbar region of the rat without obstructing the spinal canal above the site of injection. The technique can be used to demonstrate segmental analgesia and the complications occasioned by the central spread of the drug. THE PREPARATION
The catheters (tig. 1) Nylon catheters, o.d. 0.75mm and i.d. 0.25mm (Portex Ltd), were cut in 200-mm lengths. The deadspace was decreased by inserting 190 mm of a 4/0 nylon surgical suture into the lumen. The terminal 10 mm were left unfilled to allow a 30-s.w.g. needle to be connected. The deadspace was measured and was usually between 3.8 and 4.5 ftlitre, and never more than 5/ilitre. Two 5/0 surgical nylon sutures were glued to the catheter with epoxy resin, 25 mm from the inserted end and 50 mm from the exterior end. The end of the catheter to be inserted was marked at 5-mm intervals for 25mm. The catheter was flushed with air and the exterior end sealed with epoxy resin. Catheters were packed individually and sterilized with ethylene oxide. Anaesthesia Anaesthesia was induced in male Wistar rats (270-400 g in weight) by placing them in a closed © The Macmillan Press Ltd 1984
BRITISH JOURNAL OF ANAESTHESIA
406
box containing 5% halothane in oxygen. Anaesthesia was maintained via a loose-fitting, coneshaped mask through which a flow of 0.2 litre"1 min of 2.5% halothane in oxygen was passed.
dura. Occasionally, resistance was felt as the tip met the lamina of the next vertebra. The catheter was sutured to the overlying lumbar fascia which was then closed with interrupted 5/0 nylon sutures. The catheter was exteriorized through a second skin incision in the midline over the upper cervical area Surgical procedure With the rat in the prone position, the back was by tunnelling it under the skin. The cervical portion shaved and a line joining the iliac crests defined. The of the catheter was fixed to the fascia of the neck skin was incised for 4 cm in a cervical and 1 cm in a muscles with a 5/0 nylon suture and the skin incicaudal direction, in the midline from this line. The sions closed with interrupted 4/0 nylon. fascia covering the lumbar muscles was divided in the same direction as the skin incision, 1 mm lateral Injection apparatus to the midline on the side nearest to the operator. Hamilton 50-^litre and 100-nlitre gas-tight Through this opening, the lamina of the penulti- syringes were used with 28-gauge needles. The mate vertebra (usually 1 cm rostral to the iliac crests) syringe was attached to a 200-mm length of was exposed by blunt dissection. A dissecting mic- polythene tubing (0.28 mm i.d. 0.61mm o.d.), roscope was used at 16 x magnification and a 1-mm and this was joined to the implanted catheter by a hole drilled with a ball-shaped diamond drill (1 mm) double-ended 30-s.w.g. dental needle. The whole in the mid-point of the lamina. When spinal vessels assembly was sterilized with ethylene oxide. It was overlying the dura were identified through the thin- primed with the drug to be injected before being ned bone, drilling was stopped and the bone flap connected to the implanted catheter. Before the first pierced with a fine hook, bone fragments being injection the sealed tip was cut off and 5nlitre of elevated and the dura exposed. For intradural can- solution injected with a 1-mm bubble of air to nulation the dura was tensed and torn gently with uncover any leaks, and demonstrate the patency of fine-pointed (0.15-mm) forceps until CSF leaked the catheter. Subsequently, the catheter could be out and the cauda equina was seen to bulge. The left open for up to 3 months. The catheter was catheter was inserted for 10-15 mm in a cervical flushed with physiological saline after each experidirection and usually slid in easily. For extradural ment (with four times the deadspace volume cannulation the catheter was fed in a cervical direc- (20nlitre)) using a clean syringe system. Persistent tion for a similar distance outside the unopened separation of the needle tubing from the implanted
Downloaded from http://bja.oxfordjournals.org/ at New York University on May 5, 2015
FlG. 1. The catheter for implantation: 1 •* the exteriorized tip is glued with epory resin; 2— points at which sutures are glued to catheter; 3-markings at 0.5-cm intervals; 4-monofilamcnt 5/0 nylon suture with threaded round-bodied needle; 5— region where the internal filament is absent; 6= the nylon catheter with the internal filament
CHRONIC SPINAL CANNULATION IN THE RAT catheter on injection indicated that the catheter had become blocked; this could be avoided by flushing the catheter at least twice a week. EXPERIMENTAL METHODS
dose to six rats, (b) In six rats, morphine 20 mg kg" 1 was administered i.p. Twenty-five minutes later, when analgesia was well-established, naloxone 5 ulitre (0.4 mg ml"1) was injected to the intrathecal space. Evaluation of analgesia and muscle power. The extent of segmental analgesia was evaluated using the haemostat pinch test; analgesia was graded as present or absent. Muscle power was graded as normal or impaired, the latter when the affected limbs were not functioning normally. Downloaded from http://bja.oxfordjournals.org/ at New York University on May 5, 2015
Injections Injections were performed at least 24 h after recovery from surgery, recovery being defined as the ability to walk steadily, drink and feed. The rat was restrained whilst the injection system was connected, but was then free to move around its cage while injections were made. The acceptability of the position of the catheter was determined by an injection of 2% lignocaine plain (preservative free). If 35 ulitre into the intradural or 50 ulitre into the extradural space did not anaesthetize the rear limbs, the animal was excluded. This happened occasionally, especially in rats which had been catheterized for more than 4 weeks. In those cases an injection of methylene blue always showed a leak from the catheter site.
407
Histopathology. Immediately following sacrifice, 10% formalin saline solution 500 ulitre was injected through the implanted catheter. The whole spinal column was removed and fixed in the same solution for 3 days. It was then decalcified for 2 weeks. Histological sections were stained with haematoxylin and eosin. RESULTS
Evaluation of the model
Evaluation of model
Speed of onset of intradural blockade. Twenty-five microlitre of 2% lignocaine was injected into six rats over 3-4 s and the time noted until the rear limbs dragged.
Speed of onset. The onset of intradural block took 6.3±0.6s.
Equipotent volumes and doses for various levels of block. The mean volume which anaesthetized and Volumes required for equipotent intradural and ex- paralysed the rear limbs after intradural injection tradural blockade. Ten-microlitre increments of 2% were 32 /ilitre, and 46 ^litre after extradural injeclignocaine were injected every minute at 1 ulitre s~' tion ( P < 0 . 0 1 ) . Recovery occurred in a mean time in a total of 23 (11 intradural and 12 extradural) rats of 43 min after intradural and in 57 min following until both rear limbs dragged and there was no extradural injection ( P < 0 . 0 5 ) (tableI). The mean response to a pinch with a haemostat in the lumbar volume of intradural injection which anaesthetized area. Recovery of motor and sensory function was all the limbs was 115 filitre and the mean volume of timed. intradural injection which resulted in respiratory arrest was 179 ^litre (table II). Volume of intradural injection to anaesthetize all limbs. In six rats, increments of 2% lignocaine 25 ulitre were injected every minute until the rat could not support its weight; the abdomen and chest TABLEI. Relative volumes (meant ±SEM) of2% lignocaitu given by rested on the floor of the cage. Recovery to normal extradural and intradural injtaion required to anaesthetize the rear function was timed. limbs Volume of intradural injection to cause respiratory arrest. In five rats, an initial injection of 2% lignocaine 50 ulitre was followed by increments of 25 ulitre every minute until respiration ceased. Effects of morphine, (a) Preservative-free morphine 45filitre (1.5mgml~ l ) was given in a single
Intradural injection ( » - 12) Eitradural injection (n-11)
Weight (g)
2%lignocaine (ulitre)
Recovery (min)
328±11
32±3
43±3
312±7
46±2
57±7
BRITISH JOURNAL OF ANAESTHESIA
408 TABLE II. Intradural volume (mean + SEM) oflignocaine necessary (a) to anaesthetize all limbs; (b) to cause respiratory arrest Weight (g)
2%Lignocaine (filitre)
Recovery (min)
321±9
115+12
63±4
323 ±14
179 ±10
Death
Four limb anaesthesia
(n-6) Respiratory arrest (it = 5)
Histopathology. Serial sections were made of the spinal cord in three rats which had received no drugs. This revealed no reaction to the catheter (fig. 2). The hole in the lamina showed new bone formation surrounding the catheter. There was
DISCUSSION
Local anaesthetics are used widely via the extradural, or intradural, routes and neurotoxicity is sometimes suspected. In addition opioid drugs have been administered by these routes in man (Christensen and Anderson, 1982; Cooper et al., 1982; Budd, Robson and Brown, 1983). Thus far, no formal screening procedure has emerged, probably, because of difficulties in developing an acceptable animal model which allows central spread of drugs. The method described by Yaksh and Rudy (1976), although extremely valuable, has only been described in rats for cannulation of the intradural space. However, on occasions, as the cannula passed distally from the cisternal membrane, major cord damage resulted, either immediately or later (Yaksh and Rudy, 1977) possibly from movements of the head transmitted to the tip, because the catheter is fixed to the skull. Furthermore, with the cranial approach, the central complications which have been seen in man were not observed (Yaksh and
w FIG. 2. Transvene tection of the spinal cord at mid lumbar region: 1 — Intradural catheter between cauda equina nerves.
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Effects of morphine, (a) The intrathecal injection of morphine produced analgesia in the head, and all four limbs; in two rats there was no analgesia in the tail, (b) Intrathecal naloxone reversed the analgesic effects of intraperitoneal morphine in all animals; in limbs, head and tail.
minor leucocyte infiltration and giant cell formation in the muscles and subcutaneous tissues.
CHRONIC SPINAL CANNULATION IN THE RAT Imrathecal cannulation from the lumbar route
409 Intrathecal cannutauon foam cervical region lYaksh and Rudy 19771
25 rrun after intrathecal morphine 45 plitre 1 l
25 mm after morphine 20 mg kg" 1 i p
FIG. 3. Comparison of the behaviour of the lumbar cannulation model with the cervical approach of Yaksh and Rudy (1977). After cervical cannulation, analgesia did not occur in the head and nalozone failed to reverse the systemic effects of interperitoneal morphine in the head. Neither of these features are found after lumbar cannulation, although one-third of the rats failed to develop analgesia in the tail.
Rudy, 1977) suggesting that there must have been some obstruction to central spread (fig. 3). In contrast, the technique described here not only allows either extradural or intradural cannulation, but central effects of the drugs were produced readily. Respiratory arrest was induced with lignocaine and with opioids (unpublished observations), and the central effects of systemically administered morphine were antagonized by the spinal administration of naloxone (fig. 3). The model has also been used successfully to demonstrate differential sensory blockade without motor paresis using intrathecal cocaine (Bahar et al., 1984). Furthermore, neurological damage has been uncommon ( < 3% in more than 200 cannulations to date), and if it occurs it is obvious immediately. Finally, recovery appeared to be slower with Yaksh and Rudy's technique since subcutaneous fluids were infused for 24 h after surgery (Larsen and Christensen, 1982). In the method described here, full recovery occurred in most animals within 1 h. Whilst this may be a result of differences in anaesthetic technique, we believe that delay in recovery may be the result of medullary oedema. In summary, the rapidity of recovery, the low frequency of neurological damage, the demonstra-
tion of central spread of drugs and the possibility of Ijpth extradural and intradural cannulation make lumbar cannulation of value for research into the effects of drugs administered in the extradural and intradural spaces. ACKNOWLEDGEMENTS
We acknowledge in particular, the help of Mr D. Ritchie, Head of the Animal Unit; the Department of Pharmacology and the Pharmacy, University Hospital of Wales; the Department of Medical Illustration; the secretarial staff of the Department of Anaesthetics and Mrs J. Brown and Mr C. Juniper for their valuable technical assistance. REFERENCES
Bahar, M., Nunn, J. F., Rosen, M., and FleckneU, P. (1984). Differential sensory and motor blockade after spinal cocaine in the rat and marmoset. Eur. J. Anaathaiol., (in press). Orr, I. A., and Dundee, J. W. (1981). Central action of spinal opiates. Anathesiology, 55, 334. Budd, K., Robson, P. J., and Brown, P. M. (1983). The treatment of chronic pain by the use of meptazinol administered into the epidural space. Postgrad. Med. J., 59,68. Christensen, F. R., and Andersen, L. W. (1982). Adverse reaction to extradural buprenorphine. Br. J. Anaath., 54,476. Cooper, G. M., Goodman, N. W., Prys Roberts, C , Jacobson, L., and Douglas, G. (1982). Ventilatory effects of extradural diamorphine. Br. J. Anaath., 54,239. Eimerl, D., Papir-Kricheli, D., Evron, S., and Cannon, A.
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5 mm after intrathecal nakwone 5ug
BRITISH JOURNAL OF ANAESTHESIA
410
LANGDAUERNDE KANNULIERUNG DES INTRADURAL-UND EXTRADURALRAUMS BEI DER RATTE
(1983). Experimental epidural analgesia: Direct effect of morphine at the spinal level; in Advances in Pain Research and Therapy Volume 5 (eds J. J. Bonica, U. lindblom and A. Iggo), p. 487. New York: Raven Press. van den Hoogen, R. H., and Colpaert, F. C. (1981). Long-term catheterization of the lumbar epidural space in rats. Pharmacol. Biochtm. Bthav., 15, 515. Kim, K.C., and Stoelting, R. K. (1980). Effect of droperidol on the duration of analgesia and development of tolerance. Antsthesiology, 53, S219. Larsen, J., and Christensen, A. V. (1982). Subarachnoidal administration of the 5-HT uptake inhibitor citalopram points to the spinal role of 5-HT in morphine antinociception. Pain, 14, 339. Yaksh, T. L., and Rudy.T. A. (1976). Chroniccatheterisationof the spinal subarachnoid space. Physiol. Bthav., 17, 1031. (1977). Studies on the direct spinal action of narcotics in the production of analgesia in the rat. / . Pharmacol. Exp. Thtr., 202,411.
Im Tierversuch wurden therapeutische Medikamente im Eitraduralraum und im Intradurah*aum getcstet. Bei mannlichen Wistar-Ratten wurde in Narkose ein Loch in den vorletzten Lendenwirbel gebohrt, durch welches ein Katheter geschoben wurde. Der Katheter wurde dann subkutan bis in den Nacken getunnelt. NacheinemMonatgabeswederimRuckenmarknoch an den Meningen eine Reizung. Die Katheter funktionierten drci Monate lang. Die Mcthode erlaubte eine freie rostrale Ausbreitung der Medikamente. Mit 2% igem Lignocain wurde zur T Jjhmnng und Anasthesie der Hinterbeine ein intradurales Volumen von 32±3fU benStigt; extradural waren 46±2(il notig (P<0,01). Zur T,ahnning allcr Bcine waren 115±12jil notig. Atemstillstand und Tod traten-nach einem mittleren intraduralen Volumen von 179 ± 15 fU ein.
CATHETERISME CHRONIQUE DE L'ESPACE SOUS-DURAL OU PERIDURAL CHEZ LE RAT
CANULACI6N CR6NICA DEL ESPACIO INTRADURAL O EXTRADURAL EN LA RATA
ZUSAMMENFASSUNG
SUMARIO
Se elaboro una preparacion animal para ensayar agentes terapeuticos en los espacios intradurales y extradurales. Bajo anestesia, se perforo un hueco en la penultima vertebra lumbar de ratas mafhaa Wistar y se canulo el espacio apropiado. Seatraveso horadando debajo de la piel una sonda para hacerla salir al cucUo. No hubo ningiinn rcaccion meningea ni tampoco de la medula espinal despues de 1 mes. Las sondas permanecieron pervias durante 3 meses. El metodo de canulacion permiti6 la libre diseminacion de las substancias por el rostro. Al usar lignocaina liana al 2%, se necesito para la paralisis y la anestesia dc los miembros traseros un volumen intradural de 32±3nlitro: el volumen necesario de inyeccion extradural era de 46±2nlitro (P<0,01). Para la paralisis de todos los miembros, se necesito un volumen intradural de 115 ± 12(xlitro. El paro respiratorio y la muerte exigieron un volumen intradural medio de 179 ± 15 ulitro.
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RESUME
Une preparation animale a etc mise au point pour tester des agents therapcutiques dans l'espace peridural et dans l'espace sousdural. Sous anesthesie generale, un trou etait perce dans l'avantdernicre vertebre lombaire de rats Wistar males, l'espace prcetabli etait catheterise et le catheter etait tunnelisc sous la pcau pour emerger au niveau du cou. Au bout d'un mois, il n'y avait ni reaction medullaire ni reaction meningee. Les catheters restaient permcables 3 mois. La technique de catheterisme permettait une libre diffusion des agents. Avec de la lignocaine a 2%, le bloc moteur et sensitif des panes arrieres demandait un volume sousarachnoidien de 32 ±3^1 et un volume peridural de 46±2(J CP<0,01). Un bloc moteur des quatre membres demandait un volume sous-arachnoidien de 115 ± 12 fi\. L'arrct respiratoire et la mort demandait un volume sous-arachnoidien moyen de 179±15nl.